CN106929492B

CN106929492B - Tangut white spine blister H+-PPase protein gene NtVP1, encoding protein and cloning method thereof

Info

Publication number: CN106929492B
Application number: CN201710014268.4A
Authority: CN
Inventors: 杨秀艳; 李焕勇; 唐欣; 刘正祥; 张华新; 朱建峰; 成铁龙; 常二梅; 段晓波; 王文军
Original assignee: Beijing Qiji Biotechnology Co Ltd; CHINESE ACADEMY OF FORESTRY
Current assignee: Beijing Qiji Biotechnology Co Ltd; CHINESE ACADEMY OF FORESTRY
Priority date: 2016-01-15
Filing date: 2017-01-09
Publication date: 2020-03-20
Anticipated expiration: 2037-01-09
Also published as: CN106929492A

Abstract

The invention belongs to the field of molecular biology, and provides a Tangut white spine tonoplast membrane H⁺-PPase protein gene NtVP1 and its coding protein, and provides a method for cloning said gene and a method for constructing a vector comprising said gene. The gene and the protein coded by the gene play an important role in resisting salt stress of Tangut white spine, and provide a new choice for breeding new plant varieties.

Description

Tangut white spine blister H+-PPase protein gene NtVP1, encoding protein and cloning method thereof

Technical Field

The invention belongs to the field of molecular biology, and relates to gene cloning and vector construction.

Background

Vacuolar membrane H⁺Transport of inorganic pyrophosphatase (V-H)⁺PPase) is a unique proton pump that acidifies vacuoles, present in terrestrial plants and a few algae, protozoa, and bacteria. The main functions of the enzyme are as follows: (1) proton pump function: free energy and H generated by hydrolyzing inorganic pyrophosphate (PPi)⁺Proton transmembrane transporter phase coupling, hydrolysis of PPi to 2 Pi, and simultaneous H in cytoplasm⁺Pumped into the vacuole through the vacuole membrane to play the role of a proton pump and the vacuole membrane H⁺-ATPases taken together form H⁺The electrochemical gradient across the tonoplast membrane provides a driving force for the active transport of various solute (amino acid, saccharide, cation, anion and the like) molecules across the tonoplast membrane. (2) Ion regionalization function: under salt stress, plants segregate a large number of inorganic ions within the cytoplasm into vacuoles, which is one of the main strategies for plants to resist salt. During the active transport of various ions, the tonoplast and plasma membrane transporters play important roles, and the driving force of the transporters comes from H established by the tonoplast proton pump⁺Across the tonoplast membrane electrochemical gradient. This is due toShows that the expression of the tonoplast proton pump gene is increased and can be tonoplast Na⁺/H⁺The antiporter provides a stronger driving force, thereby further enhancing the salt tolerance of the plant. (3) Role in plant growth and development: vacuolar membrane H⁺PPase has a close relationship with the transport of auxin, and the interaction of the PPase and the auxin influences the growth and development of plants. (4) Participating in the biosynthesis of sucrose: sucrose in plants is converted to glucose and hexose phosphate by sucrose synthase and UDPG pyrophosphorylase, and more PPi is produced during this process, and these PPi are usually eliminated by VHP. From these results, it can be seen that vacuolar membrane VHP plays an important role in plant stress tolerance.

Tangut white thorn (Nitraria tangutorum) belongs to the genus Nitraria (Zygophylaceae) of Zygophyllaceae, is a specific halophyte in China, and is mainly distributed in northwest salinized desert. The special white spiny plants in Tanggu have developed roots, small leaves and fleshy texture, have the stress resistance of resisting wind and sand, resisting drought, resisting salt and alkali, and the like, are typical halophytes (salt ions are gathered in vacuoles), can normally grow in regions with the soil salt content of 2 percent, and are one of important group building species of desert vegetation in northwest of China. It has been found in many studies that Na⁺The division in vacuole is a necessary link for the salt tolerance of plants under high salt stress, and the applicant has already conducted previous period on the vacuole membrane Na in the Tangut white thorn⁺/H⁺The result of the successful cloning and the expression research of the antiporter gene NtNHX1 shows that the NtNHX1 gene exists in different organs of Nitraria tangutorum bobr and is induced by NaCl and the transcription level of the NtNHX1 gene is obviously increased at the early stage of salt treatment, and the results all suggest that the NtNHX1 gene plays an important role in the salt tolerance of Nitraria tangutorum bobr. As described above, the tonoplast membrane H⁺the-PPase proton pump is tonoplast membrane Na⁺/H⁺The antiporter provides driving force, so the antiporter protein can be used for treating the Tangut white spine tonoplast membrane H⁺The research of the PPase protein gene plays an important role in producing salt-tolerant plants. However, the Tangut white spine tonoplast membrane H in the prior art does not exist⁺The PPase protein gene and related reports.

Disclosure of Invention

The invention aims to provide a Tangut white spine tonoplast H⁺The PPase protein gene and the cloning method thereof, to solve the above problems in the prior art.

In a first aspect of the invention, there is provided a Thangstegia tangutica Bursa vacuole H⁺The full-length cDNA sequence of the PPase protein gene NtVP1 is shown as SEQ ID NO. 6.

Furthermore, the sequence of the coding region of the gene is shown as 106-2409 of SEQ ID NO. 6.

A second aspect of the present invention is to provide a Thangodex leucovora tonoplast H⁺The amino acid sequence of the PPase protein is shown as SEQ ID NO. 7.

The third aspect of the invention is to provide a cloned Tangut white spine tonoplast membrane H⁺-a method of the PPase protein gene NtVP1, comprising the steps of:

1) according to the vacuolar membrane H of the plant with the close relation with the Tanggute white thorn⁺-design of degenerate primer pairs for conserved regions of the PPase protein gene, preferably said degenerate primer pairs are NtVP1-F1 and NtVP 1-R1:

NtVP1-F1：CATTCGCCATTCAGG(SEQ ID NO:1)，

NtVP1-R1：ACATAGCAGCCAAAGT(SEQ ID NO:2)；

2) taking total RNA of the Nitraria tangutorum leaves as a template, and obtaining a cDNA fragment by using the degenerate primer pair of the step 1) through an RT-PCR method; preferably, the cDNA fragment is 1156bp in length (SEQ ID NO: 3);

3) designing UPM primers, 3'RACE primers and 5' RACE primers according to the cDNA fragments obtained in the step 2), and obtaining a full-length cDNA sequence through nested amplification, wherein the nucleotide sequence of the full-length cDNA is shown as SEQ ID NO. 6; preferably, the primer sequences are as follows:

UPM(SEQ ID NO:10)：

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT；

3’RACE：TGCCAATCCAGTGGCAATAGTGCTGA(SEQ ID NO:4)；

5’RACE：ACAACTGCTGCCATCGGAAAGGGATT(SEQ ID NO:5)。

preferably, the plant related to Tangut white spine is Tyrophora ovata.

The fourth aspect of the invention provides an expression vector or an expression strain, wherein the expression vector or the expression strain comprises the Tangut white spine tonoplast membrane H shown as SEQ ID NO. 6⁺The cDNA sequence of the PPase protein gene NtVP1 or the coding sequence thereof. Preferably, the expression vector is a vector carrying a GFP gene, and the expression strain is Agrobacterium.

The fifth aspect of the invention provides a construction method of an expression vector, wherein the expression vector comprises the Bursa of Tangut white spine vacuolar membrane H shown in SEQ ID NO:6⁺-the cDNA sequence of the gene NtVP1 of the PPase protein or the sequence of its coding region, comprising: 1) xba I and Kpn I enzyme cutting sites are respectively added at two ends of a cDNA sequence of the NtVP1 gene or a coding region sequence thereof and are connected with a T vector to form a T-NtVP1 vector; 2) the T-NtVP1 vector, pCG-GFP and pSN1301 were digested with Xba I and Kpn I, respectively, and recovered; 3) the gene fragment cleaved from the T-NtVP1 vector was ligated to the cleaved fragments of the pCG-GFP and pSN1301 vectors.

The sixth aspect of the invention provides a Tangut white spine blister H⁺A method for transfecting a plant with the PPase protein gene NtVP1, which comprises transfecting a plant with the Bursa of Tangut white spine membrane H as shown in SEQ ID NO:6⁺The cDNA sequence of the PPase protein gene NtVP1 or the vector of the coding region sequence thereof was transformed into Arabidopsis thaliana.

The seventh aspect of the present invention is to provide the Tangut white spine tonoplast H of the present invention⁺Application of the PPase protein gene NtVP1 in preparation or cultivation of transgenic plants.

The invention utilizes the vacuole membrane H of different species which are close to the relativity of the Tanggute white spine⁺Degenerate primer is designed in conserved region of PPase protein gene and first cloned to the Langchinus Tangut white spine vacuolar membrane H by means of RT-PCR and RACE⁺The gene NtVP1 of the PPase protein, and the functional analysis and the research of the transcription level of the gene under the conditions of non-saline environment and high salt stress are carried out respectively, thereby defining the resistance of the gene and the encoded enzyme protein to salt in Tangut white spine at the molecular levelThe function in stress provides a new choice for breeding new plant varieties.

Drawings

FIG. 1 shows the Tangut white spine tonoplast H⁺The cDNA sequence and the amino acid sequence of PPase (VP 1).

FIG. 2 shows tonoplast membrane H from different species⁺-PPase (VP1) phylogenetic tree analysis graph.

FIG. 3 shows the Tangut white spine tonoplast H⁺-bioinformatics analysis map of PPase (NtVP 1): A. VP1 protein domain analysis; B. analyzing the hydrophobicity of the VP1 protein; C. analyzing a VP1 protein transmembrane region; D. VP1 protein secondary structure prediction.

FIG. 4 shows the Tangut white spine tonoplast H⁺qRT-PCR expression profile of the PPase protein gene (NtVP1) in different organs; A. relative expression levels of the NtVP1 gene in different organs; B. relative expression quantity of NtVP1 gene in Tangut white spine leaves under NaCl stress.

FIG. 5 shows a schematic diagram of the pCG-GFP vector structure.

FIG. 6 shows a schematic diagram of the pSN1301 vector structure.

FIG. 7 shows an electrophoretogram of NitVP 1 gene from Nitraria tangutorum bobr which has been ligated to T vector; wherein, Marker is DL2000 plus; 1-6 are 6 detected E.coli clones, 2, 4, 5, 6 are 4 positive clones.

FIG. 8 shows an electrophoretogram of a gene fragment of NtVP1 obtained by double digestion of the T-NtVP1 vector with Xba I and Kpn I.

FIG. 9 shows an electrophoretogram of open circular plasmid obtained after double digestion with Xba I and Kpn I on pCG-GFP and pSN1301 vectors.

FIG. 10 shows an electrophoretogram of positive plaques transformed with pCG-35S:: NtVP1-GFP and pSN1301-35S:: NtVP1, using primers T1 and T2 specific for the NtVP1 gene. M represents DL2000 plus; the VP1-pCG at the left is pCG-35S, and the detection result of the NtVP1-GFP positive clone is obtained; the right VP1-pSN1301 are pSN1301-35S:, the result of the detection of the NtVP1 positive clone.

FIG. 11 shows a graph comparing chlorophyll, proline and malondialdehyde content and SOD activity of Arabidopsis plants at the T3 generation and wild type after 30mmol/L NaCl stress; A. comparison of chlorophyll content; B. a graph comparing proline content; C. a malondialdehyde content comparison graph; D. SOD activity is shown in the figure.

Detailed Description

The present invention is further illustrated by the following specific examples, but the scope of the invention is not limited thereto. Unless otherwise specified, the reagents, instruments, consumables, and the like used in the following examples are all reagents, instruments, and consumables that are common in the art and are available from conventional chemical stores or suppliers. The methods used in the following examples are conventional in the art, unless otherwise specified; the skilled person will be able to know clearly how to carry out the experiments from the experimental description.

In the present application, NtVP1 denotes the Bursa membrane H of Tangut white⁺PPase protein gene, NtVP1 for Nitraria tangutorum bobr Echinacea H⁺-a PPase protein.

The first embodiment is as follows: tangut white spine blister H⁺Cloning of the PPase protein Gene (NtVP1)

A pair of degenerate VP1-F1(SEQ ID NO:1) and NtVP1-R1(SEQ ID NO:2) primers are designed in the amino acid conserved region according to a multiple sequence alignment of the cloned genes of 10 species (Gossypium hirsutum ADN96173.1), grape (vitas vinifera XP _002273207.1), Populus trichocarpa (Populus trichocarpa XP _006381091.1), Arabidopsis thaliana (NP _173021.1), Salicornia europaea (AEI17666.1), Salicornia javanica (Kalidium folicum ABK91685.1), Oryza sativa (BAA31523.1), Suaeda cornicifolia ADQ00196.1), Halostachys chinensis ABO45933.1, and Tyveda rex (ABU 92563.1)). Total RNA from Nitraria tangutorum leaves (RNAasso Plus from Takara) was extracted, and a cDNA fragment (1156bp) of a conserved region was amplified from the total RNA of Nitraria tangutorum by RT-PCR (SEQ ID NO: 3). The RT-PCR method is as follows:

mu.l of total RNA, 1. mu.l of 50. mu.M oligo (Dt)₂₀Mu.l of a 10mM dNTP mixture (all on ice) was added to a 0.2ml RNase-free centrifuge tube, gently mixed, immediately denatured at 65 ℃ for 5min, then immediately placed on ice for at least 1min, followed by mixing of the following components as followsAnd (4) homogenizing.

Add 10. mu.l of the mixture to a centrifuge tube containing the RNA/primer mixture, gently beat and mix well, and immediately 50 ℃ 50min, 85 ℃ 5 min. Immediately after the above procedure, the tube was placed on ice for 1 minute, and after a slight centrifugation, 1. mu.l of RNase H was added to the tube and incubated at 37 ℃ for 20 min. First strand cDNA was synthesized. The synthesized cDNA was used as a template, and conserved region PCR amplification and sequencing were performed using primers NtVP1-F1(SEQ ID NO:1) and NtVP1-R1(SEQ ID NO: 2).

Based on the cDNA fragments of the conserved regions obtained by cloning, UPM primers (SEQ ID NO:10), terminal amplification primers 5'RACE (SEQ ID NO:4) and 3' RACE (SEQ ID NO:5) were designed and subjected to the rapid amplification of cDNA terminals (RACE) (SMARTer from Clontech corporation)^TMRACE cDNA Amplification Kit), and amplifying total RNA of Nitraria tangutorum bobr leaves to obtain Nitraria tangutorum bobr vacuole membrane H⁺The full-length cDNA sequence of the PPase protein gene. The full-length cDNA was 2810bp in length by sequencing (SEQ ID NO: 6).

The resulting full-length cDNA sequence was submitted to NCBI for alignment and found to be associated with type I H of plants such as Gossypium hirsutum, Salicornia europaea, Vitis vinifera and Thellungiella salginea⁺The PPase protein gene has homology of more than 80%, and the full-length cDNA sequence of the gene is named as NtVP 1.

Translating the cDNA sequence into amino acid by using NCBI to obtain the Tangut white spine tonoplast membrane H⁺The protein of PPase is named as NtVP1, and the amino acid sequence of the protein is shown as SEQ ID NO. 7.

Example two: vacuolar membranes H of different species⁺Comparison of the PPase protein sequences

Selecting 10 vacuolar membranes H from different plants from GenBank⁺Amino acid sequences of the PPase protein, cotton (Gossypium hirsutum ADN96173.1), grape (Vitis vinifera XP-002273207.1), Populus trichocarpa XP-006381091.1, respectively,Arabidopsis thaliana (NP-173021.1), Salicornia europaea (AEI17666.1), Salicornia graticus (Kalidium receptacle ABK91685.1), Oryza sativa (BAA31523.1), Suaeda corniculata (Suaeda cornicifolia ADQ00196.1), Halostachys caspica ABO45933.1, and Tyrophora rex (ABU92563.1), and these 10 proteins were compared with the NtVP1 protein of the present invention in multiplex using DNMAN software. The results showed that the amino acid sequence of NtVP1 was identical to that of the cloned upland cotton H⁺The homology of PPase protein reaches 93 percent and is similar to the Arabidopsis thaliana vacuole membrane H⁺The homology of the PPase protein AtVP1 is 89.29%, and the homology is with the Arabidopsis thaliana vacuolar membrane H⁺The homology of the PPase protein AtVP2 is only 33.53% (AAF 31163.1). In addition, NtVP1 and other plant vacuoles H⁺PPase protein also has higher homology (FIG. 1). As can be seen in FIG. 1, the protein contains 3 conserved regions (CS1, CS2 and CS3), wherein the CS1 conserved region contains DVGADLVGKVE sequences, and (E/X) (X)₇KXE configuration. This configuration is in the vacuolar membrane H of many plants⁺Transport inorganic pyrophosphatases are highly conserved and may be present as substrate binding sites for the hydrolysis of pyrophosphate (PPi). In addition, it was also found from FIG. 1 that the protein contains the ` EYYTS ` motif, which is involved in the hydrolysis of pyrophosphate and H⁺The coupling of transport and its presence in most other vacuolar proton pump pyrophosphate hydrolases (V-PPases).

Example three: tonoplast membrane H from different species⁺Phylogenetic tree of PPase proteins

In vacuolar membrane H⁺On the basis of the alignment of PPase protein sequences, the 10 tonoplast membranes H from different plants are aligned by using ClustalX software⁺The amino acid sequences of the PPase proteins (Gossypium hirsutum ADN96173.1), Vitis vinifera (Vitis vinifera XP _002273207.1), Vitis vinifera (CAO41672.1), Populus trichocarpa XP _006381091.1, Arabidopsis thaliana (NP _173021.1) and (AAF31163.1), Salicornia herbacea (AEI17666.1), Salicornia herbacea (Kalidium foliatum ABK91685.1), Oryza sativa (BAA31523.1), Suaeda corniculata (Suaeda cornicuta AD00196.1), Halostachys chinensis (Halostachys caspica ABO45933.1), and Tyrium king (ABU92563.1)) and the NtVP1 proteins evolved according to the present invention were subjected to multiple sequence matching arrangement, and then the construction of the phylogenetic tree was carried out using MEGA5.1 softwareThe method is carried out by 1000 bootstrap analyses according to the Neighbor-join method by the Poisson distance method. The results (FIG. 2) show that the Bursa of Tangut white spine H⁺The genetic distance between the-PPase protein gene NtVP1 and the upland cotton GhVP1, the Arabidopsis AtVP1, the grape VvVP1 and the like belonging to the I type is relatively short, and the genetic distance between the-PPase protein gene NtVP1 and the Arabidopsis AtVP2 and the grape VvVP2 belonging to the II type is relatively long.

Example four: tangut white spine blister H⁺Sequence analysis of the PPase protein Gene NtVP1

The protein sequence coded by the full-length NtVP1cDNA sequence is a protein with 768 amino acids. The molecular weight is 80.5kDa according to the sequence, and the isoelectric point is 5.33; the protein was found to have more hydrophobic amino acids distributed uniformly throughout the peptide chain than hydrophilic amino acids using hydrophobicity analysis software (http:// web. expasy. org/cgi-bin/protscale. pl) (a in fig. 3). Thus, the entire polypeptide chain appeared hydrophobic, indicating that the NtVP1 protein conforms to the characteristics of a membrane protein, belonging to a hydrophobic protein (B in fig. 3). Transmembrane analysis using TMHMM software revealed that NtVP1 contains 14 transmembrane regions (C in FIG. 3), which is in contrast to tonoplast membrane H in other species⁺The transmembrane domain of the PPase protein is identical. The structure of NtVP1 protein was analyzed by Swiss-Model (http:// swisssmall. expasy. org /), and it was found to have up to 90.21% similarity to the sequence of the 4a01.1.A template, belonging to the proton pyrophosphatase Model (D in FIG. 3).

Example five: expression analysis of Nitraria tangutorum bobr NtVP1 under NaCl high-salt stress

Total RNA (columnar plant RNAout kit of Beijing Tianenzze Gene Co., Ltd.) was extracted from young leaves, young stems and young roots of Nitraria tangutorum bobr, respectively, and 500ng was used as a template and synthesized by reverse transcription to form 1 st strand cDNA (SuperScript IIIReverse Transcriptase). Based on the full-length sequence of the NtVP1cDNA, 1 pair of specific primers NtVP1-F8(SEQ NO8) and NtVP1-R8(SEQ NO 9) are designed, and a segment with the length of 207bp is obtained through amplification (SEQ ID NO: 13). The expression of NtVP1 in different organs of Nitraria tangutorum bobr under normal growth conditions is detected by using an Actin gene (Genbank: AB617805.1) of Nitraria tangutorum bobr as an internal reference and adopting a fluorescent quantitative PCR method. The experimental conditions of the fluorescent quantitative PCR are as follows: pre-denaturation at 95 ℃ for 10min, then at 95 ℃ for 15s, at 60 ℃ for 1min, for 40 cycles. The fluorescent dye is SYBR Green I.

The results show that NtVP1 is expressed in different organs and the expression level in leaf blades>Amount of expression in root>Expression level in stem (A in FIG. 4). In order to investigate whether the VP1 gene of the Nitraria tangutorum bobr is induced and regulated by salt, 200 mmol.L is further detected^-1Expression level of NtVP1 in Nitraria tangutorum leaves after NaCl stress for 1, 3, 6, 12, 24 and 48 h. The specific method comprises the following steps: seeds of nitraria tangutorum were sown in vermiculite: the turf is 3: 1(V/V) in a mixed medium, and culturing in a greenhouse. Selecting 3-month-old seedlings which grow normally and have consistent sizes, culturing with tap water, and changing water 1 time every 4 days during the culture period. Hydroponic culture for about 1 week with 0(CK) and 200mmol "L^-1Seedlings were treated with NaCl for 1, 3, 6, 12, 24, 48h, 3 replicates each treatment, 3 replicates each, for a total of 108. Uniformly collecting tender leaf tissues at 10: 00-11: 00 in the morning. The results show that the expression level of the NitzVP 1 gene of Nitzkola tangutorum bobr is continuously increased in leaves with the prolongation of the salt stress time, the expression level is highest when the Nitzkola tangutorum bobr is stressed for 12 hours and then gradually weakened (B in figure 4), and the results show that the NitzVP 1 gene may play an important role in the Nitzkola tangutorum bobr salt tolerance mechanism.

Example six: construction of Nitraria tangutorum bobr NtVP1 gene expression vector

Firstly, determining a pCG-GFP vector (figure 5) (Jun organism) and a pSN1301 vector (figure 6) (Jun organism), adding Xba I and Kpn I enzyme cutting sites at two ends of a gene, and connecting the gene to a T vector (Transgen, CT11-01, http:// www.transgen.com.cn/products/63. html); the insertion site fragment was amplified using gene-specific primers T1 and T2, and a band containing the NtVP1 gene was obtained by PCR examination, confirming that the NtVP1 gene had been ligated to the T vector (FIG. 7). Wherein, the primer sequences of T1 and T2 are respectively: t1: 5'-GTTTGCCTTCGTCCTTCA-3' (SEQ ID NO: 11); t2: 5'-CATTGTCCCATGCACCTC-3' (SEQ ID NO: 12).

The T-NtVP1 vector, pCG-GFP and pSN1301 were digested with Xba I and Kpn I, respectively, to obtain the NtVP1 gene fragment (FIG. 8) and pSN1301 and pCG-GFP open loop vectors (FIG. 9), the NtVP1 gene was ligated to pSN1301 and pCG-GFP, E.coli was transformed, and then the transformants were PCR-tested to obtain a test map (FIG. 10), confirming that the vector construction was complete and a positive clone was obtained. The vector pSN1301 ligated to the NtVP1 gene was sequenced, and the sequences flanking the gene were determined to be the sequence of the vector. It was confirmed that the NtVP1 gene had been ligated to an expression vector.

Example seven: nitraria tangutorum bobr NtVP1 gene transformation

The two binary vectors constructed in example six were transformed into Agrobacterium C58, cultured, inoculated into 5ml LB liquid medium containing antibiotics (rifampicin 20mg/L, kanamycin 50mg/L), and shake-cultured at 28 ℃ for 2 days. 1ml of the cultured Agrobacterium was transferred to 100ml of LB liquid medium containing antibiotics and the shake culture was continued at 28 ℃ for 24 hours. Pouring the bacterial liquid into a centrifuge tube, centrifuging at 6000rpm/min at room temperature for 10 minutes, and then pouring out the supernatant. The pellet was resuspended in 200ml of a staining solution (10% sucrose, 0.02% silwet) to form a uniform Agrobacterium suspension (OD)₆₀₀0.8) and the agrobacterium suspension is transferred to an open vessel (500ml beaker). Selecting healthy arabidopsis thaliana at the initial fruiting stage, reversely buckling the arabidopsis thaliana above a container containing the agrobacterium suspension, only immersing the whole inflorescence into the agrobacterium suspension for about 20-30 seconds, taking down the pot, and transversely placing the pot in a dark box for about 24 hours. And after 24 hours, placing the treated arabidopsis thaliana plant under the illumination condition of 22-25 ℃ for normal growth, and harvesting mature seeds after three weeks.

Example eight: salt stress validation analysis of Arabidopsis thaliana

The arabidopsis thaliana seeds harvested in the seventh embodiment are sown in 1/2MS culture medium containing 25mg/L hygromycin for screening, when two true leaves grow out, the arabidopsis thaliana seeds are transplanted in a culture medium containing turfy soil and vermiculite (turfy soil: perlite: 1), arabidopsis thaliana plants are placed under the condition of illumination at 22-25 ℃ for normal growth, and mature seeds are harvested after three weeks. The above operation is repeated until T3 generation seeds (TR) are collected. And (3) respectively sowing and culturing T3 generation arabidopsis seeds (TR) and wild type arabidopsis seeds (WT), and carrying out NaCl stress treatment two weeks after transplanting. A control group (0) and a treatment group (1) were provided, each in triplicate, wherein the treatment was 30mmol/L NaCl. And (4) collecting leaves after stress treatment for 4d, immediately freezing the leaves by liquid nitrogen, and respectively measuring the chlorophyll content, the proline content, the malondialdehyde content and the SOD activity change.

The results show that the chlorophyll content of the T3 generation plants is 0.0334mg/g in the control (TR0) and 0.0338mg/g after salt stress treatment (TR1), and no significant difference is generated (p is more than 0.05); malondialdehyde content was 0.0025umol/g in the control (TR0), and became 0.0031umol/g after salt stress treatment (TR1), with no significant difference (p > 0.05); the proline content of T3 generation plants is 33.38ug/gFw (TR0) and 105.47ug/gFw (TR1) before and after salt stress respectively, the content is obviously increased after the salt stress, and the obvious difference exists (p is less than 0.05); SOD activity was 7.445U/gFW in the control (TR0), changed to 17.056U/gFW after salt stress treatment (TR1), and was significantly increased with a significant difference (p <0.05) after salt stress treatment (FIG. 11).

After salt stress of wild plants, the chlorophyll content is changed from 0.039mg/g (WT0) to 0.029mg/g (WT1), and a significant difference exists (p is less than 0.05); the malonaldehyde content increased from 0.0034umol/g (WT0) to 0.0048umol/g (WT1), with a significant difference (p < 0.05); the proline content was changed from 55.43ug/gFw (WT0) to 68.52ug/gFw (WT1), with no significant difference (p > 0.05); SOD activity was 5.42U/gFW (WT0) in the control group and 6.77U/gFW (WT1) after salt stress treatment, with no significant difference (p >0.05) (FIG. 11).

The result shows that salt stress promotes the synthesis of proline which is a T3 generation plant osmoregulation substance, the content of malondialdehyde is not increased remarkably, and the synthesis and degradation of chlorophyll are not influenced remarkably, which indicates that a membrane system is more stable; meanwhile, the SOD activity is obviously increased, which proves that the capability of eliminating superoxide anion free radicals is enhanced. The experiments prove that the salt tolerance of the T3 generation plant is obviously improved by the successful transformation of the NtVP1 gene in an Arabidopsis plant, namely the NtVP1 gene has an active effect on the aspect of improving the salt tolerance of the plant.

Sequence listing

SEQ ID NO:1：CATTCGCCATTCAGG

SEQ ID NO:2：ACATAGCAGCCAAACT

SEQ ID NO:3：

GATGATTGGGAGGGTCTTTTGAGGCTATCCRAGCATTTGGGCTGACGGGATCGACTACGCATGCTATGGGACGACCGGGCGGCGGTCTCTATCCGATTGCTGCTGATGTTGGTGCTGATCTTGTGGGCAAGGTCGAGAGAAACATTCCAGAAGACGAGCCAAGAAACCCTGCTGTCATTGCTGACAACGTTGGTGACAATGTTAGGGACGTTGCTCGCATGGGCTCAGATCTTTTCGGTTCATATGCCGGGTCATCTTGTGCTGCGCTCGTAGTTGCATCCATTTCATCCTTTGGAATCAACCATGACTTCACTGCCATGTTGTATCCTCTGCTCATCAGTTCGATGGGTATCCTTGTTTGTTTGATCACAACTCTCTTTGCCACTGATATCTTTGAGATCAAGGCTGTTAAAGAGATCGAGCCAGCATTGAAGAAGCAGCTTATCATCTCTACTATTCTTATGACTGTTGGAATTGCAATTGTTTCATGGGTTGGTTTGCCTTCGTCCTTCACAATCTACAATTTTGGGACTCAGAAGGTTGTCAAGAACTGGGAACTTTTCTTGTGTGTGGGTGTTGGTCTTTGGGCTGGACTCATCATTGGATTTGTGACCGAGTACTATACTAGCAACGCATACAGCCCTGTACAAGATGTTGCTGACTCCTGCAGAACAGGAGCTGCCACCAATGTTATCTTTGGTCTTGCTTTGGGATACAAATCTGTCATCATTCCAATTTTTGCCATTGCTATCAGTATTTTTGTCAGTTTTAGTTTGGCTGCTATGTATGGCATTGCAGTGGCTGCCCTTGGTATGCTCAGCACTATTGCCACTGGATTGGCAATTGATGCCTACGGTCCCATCAGTGACAATGCTGGAGGCATTGCTGAGATGGCTGGCATGAGTCACCGCATCCGTGAGAGGACTGATGCCCTTGATGCAGCTGGAAACACAACTGCTGCCATCGGAAAGGGATTTGCAATTGGATCAGCAGCCCTTGTCTCTTTGGCTCTGTTTGGTGCTTTTGTTAGCCGTGCAGCAATCTCAACAGTTGATGTCTTGACTCCAAAGGTGTTCATCGGTTTGATTGTTGGTGCTATGCTCCCGTACTGGTTCTCGGCCACATGACCATCGGAAGAGTGTNGGAAGTGCAATCT

SEQ ID NO:4：TGCCAATCCAGTGGCAATAGTGCTGA

SEQ ID NO:5：ACAACTGCTGCCATCGGAAAGGGATT

SEQ ID NO:6：

ACATGGGGAAACATCTTCACTTGAAAATACTTCCCTCTCCTCTGTCTTCTGCCTCTTCTCTTTTTCTTTTTTTTTTTTTTCGTTGTTTTTCTGGTTTTGGTAGGTATGGGAGTGGCGTTGCTGTCCGAGCTGGCGACGGAGATACTGGTTCCGGTCTGTGCCGTGATCGGTATCGTGTTCTCGCTCGTTCAGTGGTACCTCGTCTCGCGCGTGTCGCTCACGCACGACCGGTCGGCCGGGAACAACAACAACAACAAGAAGAATGGATTCAACGATTATTTGATCGAGGAAGAGGAAGGAATTAATGACCAGAGCGTCGTGACCAAGTGTGCTGAAATTCAGAACGCTATTTCTGAAGGTGCAACATCCTTTCTTTTCACTGAATATCAGTATGTTGGGATCTTCATGGTTGCTTTTGCAATCTTGATTTTCCTCTTCCTGGGTTCTGTGGAGGGCTTCAGCACAAAGAGCCAGCAATGTACTTACGATAAAACAAGGACGTGCAAGCCTGCACTTGCCACTGCTATCTTCAGCACAGTAGCATTTGTGCTTGGTGGCGTCACATCTGTCCTTTCTGGCTTCCTTGGGATGAAAATTGCTACTTATGCAAATGCCAGAACTACCCTGGAAGCAAGAAGGGGTGTCGGAAAGGCTTTTATTACTGCATTTAGGTCTGGTGCAGTAATGGGCTTCCTCCTTGCAGCAAATGGTCTCTTGGTGCTTTACATTGCTATCAATCTCTTTAAGTTGTACTATGGTGATGACTGGGAAGGCCTATTTGAGGCTATTACTGGATACGGTCTTGGGGGTTCTTCAATGGCTCTCTTTGGACGAGTGGGTGGTGGTATCTATACCAAGGCTGCTGATGTTGGTGCTGATCTTGTGGGCAAGGTCGAGAGAAACATTCCAGAAGACGATCCAAGAAACCCTGCTGTCATTGCTGACAACGTTGGTGACAATGTTGGGGACATTGCTGGCATGGGCTCGGATCTTTTTGGTTCTTATGCTGAGTCATCCTGTGCTGCGCTTGTTGTTGCATCCATTTCATCCTTTGGAATCAACCATGACTTCACTGCCATGTTGTATCCTCTGCTCATCAGTTCGATGGGTATCCTTGTTTGTTTGATCACAACTCTCTTTGCCACTGATATCTTTGAGATCAAGGCTGTTAAAGAGATCGAGCCAGCATTGAAGAAGCAGCTTATCATCTCTACTATTCTTATGACTGTTGGAATTGCAATTGTTTCATGGGTTGGTTTGCCTTCGTCCTTCACAACCTACAATTTTGGGACTCAGAAGGTTGTCAAGAACTGGGAACTTTTCTTGTGTGTGGGTGTTGGTCTTTGGGCTGGACTCATCATTGGATTTGTGACCGAGTACTATACTAGCAATGCATACAGCCCTGTACAA GATGTTGCTGACTCCTGCAGAACAGGAGCTGCCACCAATGTTATCTTTGGTCTTGCTTTGGGATACAAATCTGTTATCATTCCAATTTTTGCCATTGCTATCAGTATTTTTGTCAGTTTTAGTTTGGCTGCTATGTATGGCATTGCAGTGGCTGCCCTTGGTATGCTCAGCACTATTGCCACTGGATTGGCAATTGATGCCTACGGTCCCATCAGTGACAATGCTGGAGGCATTGCTGAGATGGCTGGCATGAGTCATCGCATCCGTGAGAGGACTGATGCCCTTGATGCAGCTGGAAACACAACTGCTGCCATCGGAAAGGGATTTGCAATTGGATCAGCAGCCCTTGTCTCTTTGGCACTGTTTGGTGCTTTTGTTAGCCGTGCAGCAATCTCAACAGTTGATGTCTTGACTCCAAAGGTGTTCATCGGTTTGATCGTTGGTGCTATGCTTCCGTACTGGTTCTCTGCCATGACCATGAAGAGTGCAGGAAGTGCTGCATTGAAAATGGTTGAGGAGGTTCGCAGGCAGTTCAACACCATTCCTGGCCTCATGGAGGGCACTGCCAAGCCTGATTACGCTAACTGTGTCAAGATCTCTACTGATGCTTCCATCAAGGAGATGATTCCTCCTGGTGCTCTTGTCATGCTCACACCCCTCATCGTCGGAACCTTCTTCGGTGTGGAAACCCTCTCTGGTGTTCTTGCTGGCTCTCTTGTATCTGGTGTTCAGATCGCAATATCTGCATCAAACACTGGAGGTGCATGGGACAATGCCAAGAAGTACATTGAGGCAGGTGCTTCTGAGCACGCAAGGACCCTTGGACCCAAAGGGTCAGAGCCACACAAGGCAGCTGTGATCGGTGACACCATCGGAGACCCACTCAAGGACACGTCGGGGCCATCACTGAACATCCTTATCAAGCTTATGGCCGTAGAATCGCTTGTTTTTGCTCCCTTTTTCGCCACCCACGGTGCCTTTCTTTTCAAAATTTTTTGAAGAAAGTTAAATAATGACCAGAAGAAGGGGGGGAAGGGAGGGGGTTTGTTAAGCTAGTTTTATTTAGGGAAGTTAAAATACTATTTTTGATTTTGAGGAGGGATGAGGAGGAGGATTTGGACACCAATGGAGATTGGGGTAAAACAAAAATTTGATGGGATGGGATGTGTGAACCAAAATTGGAGGCATAAATATGGGGCATCAGGACTTGGGTGGGTTGGGGGTATGTTCTGGTCCTTTTTAATTTTAATTTTTGGTTTTTTTTTTTTTTGTTTCATGTAGGGGGTAGAACAATTTTCCCTTTTTTATTTTATTTTAACATTTGTACTGTTCATCGTTTAATCAAGAAAAGACCATTTGGATTTTTTACCAAAAAAAAAAAAAAAAAAAAAAAAGTACTT

SEQ ID NO:7：

MGVALLSELATEILVPVCAVIGIVFSLVQWYLVSRVSLTHDRSAGNNNNNKKNGFNDYLIEEEEGINDQSVVTKCAEIQNAISEGATSFLFTEYQYVGIFMVAFAILIFLFLGSVEGFSTKSQQCTYDKTRTCKPALATAIFSTVAFVLGGVTSVLSGFLGMKIATYANARTTLEARRGVGKAFITAFRSGAVMGFLLAANGLLVLYIAINLFKLYYGDDWEGLFEAITGYGLGGSSMALFGRVGGGIYTKAADVGADLVGKVERNIPEDDPRNPAVIADNVGDNVGDIAGMGSDLFGSYAESSCAALVVASISSFGINHDFTAMLYPLLISSMGILVCLITTLFATDIFEIKAVKEIEPALKKQLIISTILMTVGIAIVSWVGLPSSFTTYNFGTQKVVKNWELFLCVGVGLWAGLIIGFVTEYYTSNAYSPVQDVADSCRTGAATNVIFGLALGYKSVIIPIFAIAISIFVSFSLAAMYGIAVAALGMLSTIATGLAIDAYGPISDNAGGIAEMAGMSHRIRERTDALDAAGNTTAAIGKGFAIGSAALVSLALFGAFVSRAAISTVDVLTPKVFIGLIVGAMLPYWFSAMTMKSAGSAALKMVEEVRRQFNTIPGLMEGTAKPDYANCVKISTDASIKEMIPPGALVMLTPLIVGTFFGVETLSGVLAGSLVSGVQIAISASNTGGAWDNAKKYIEAGASEHARTLGPKGSEPHKAAVIGDTIGDPLKDTSGPSLNILIKLMAVESLVFAPFFATHGAFLFKIF

SEQ ID NO:8：GTTCGCAGGCAGTTCAACACCATTC

SEQ ID NO:9：ACCAGATACAAGAGAGCCAGCAAGA

SEQ ID NO:10：

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT

SEQ ID NO:11：GTTTGCCTTCGTCCTTCA

SEQ ID NO:12：CATTGTCCCATGCACCTC

SEQ ID NO:13：

GTTCGCAGGCAGTTCAACACCATTCCTGGCCTCATGGAGGGCACTGCCAAGCCTGATTACGCTAACTGTGTCAAGATCTCTACTGATGCTTCCATCAAGGAGATGATTCCTCCTGGTGCTCTTGTCATGCTCACACCCCTCATCGTCGGAACCTTCTTCGGTGTGGAAACCCTCTCTGGTGTTCTTGCTGGCTCTCTTGTATCTGGTGTTCAGATCGCAATATCTGCATCAAACACTGGAGGTGCATGGGACAATGCCAAGAAGTACATTGAGGCAGGTGCTTCTGAGCACGCAAGGACCCTTGGACCCAAAGGGTCAGAGCCACACAAGGCAGCTGTGATCGGTGAC ACCATCGGAGACCCACTCAAGGACACGTCGGGGCCATCACTGAACATCCTTATCAAGCTTATGGCCGTAGAATCGCTTGTTTTTGCTCCCTTTTTCGCCACCCACGGTGCCTTTCTTTTCAAAATTTTTTGAAGA

Sequence listing

<110> scientific research institute of forestry in China

Beijing Qi Xijiu biotech Co Ltd

<120> Thangstegia tangutica H + -PPase protein gene NtVP1, encoding protein thereof and cloning method

<130>TD1612546F

<160>13

<170>PatentIn version 3.5

<210>1

<211>15

<212>DNA

<213> Artificial sequence

<220>

<223> degenerate primer NtVP1-F1

<400>1

cattcgccat tcagg 15

<210>2

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> degenerate primer NtVP1-R1

<400>2

acatagcagc caaagt 16

<210>3

<211>1156

<212>DNA

<213> Tanggute white thorn

<400>3

gatgattggg agggtctttt gaggctatcc ragcatttgg gctgacggga tcgactacgc 60

atgctatggg acgaccgggc ggcggtctct atccgattgc tgctgatgtt ggtgctgatc 120

ttgtgggcaa ggtcgagaga aacattccag aagacgagcc aagaaaccct gctgtcattg 180

ctgacaacgt tggtgacaat gttagggacg ttgctcgcat gggctcagat cttttcggtt 240

catatgccgg gtcatcttgt gctgcgctcg tagttgcatc catttcatcc tttggaatca 300

accatgactt cactgccatg ttgtatcctc tgctcatcag ttcgatgggt atccttgttt 360

gtttgatcac aactctcttt gccactgata tctttgagat caaggctgtt aaagagatcg 420

agccagcatt gaagaagcag cttatcatct ctactattct tatgactgtt ggaattgcaa 480

ttgtttcatg ggttggtttg ccttcgtcct tcacaatcta caattttggg actcagaagg 540

ttgtcaagaa ctgggaactt ttcttgtgtg tgggtgttgg tctttgggct ggactcatca 600

ttggatttgt gaccgagtac tatactagca acgcatacag ccctgtacaa gatgttgctg 660

actcctgcag aacaggagct gccaccaatg ttatctttgg tcttgctttg ggatacaaat 720

ctgtcatcat tccaattttt gccattgcta tcagtatttt tgtcagtttt agtttggctg 780

ctatgtatgg cattgcagtg gctgcccttg gtatgctcag cactattgcc actggattgg 840

caattgatgc ctacggtccc atcagtgaca atgctggagg cattgctgag atggctggca 900

tgagtcaccg catccgtgag aggactgatg cccttgatgc agctggaaac acaactgctg 960

ccatcggaaa gggatttgca attggatcag cagcccttgt ctctttggct ctgtttggtg 1020

cttttgttag ccgtgcagca atctcaacag ttgatgtctt gactccaaag gtgttcatcg 1080

gtttgattgt tggtgctatg ctcccgtact ggttctcggc cacatgacca tcggaagagt 1140

gtnggaagtg caatct 1156

<210>4

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223>3' RACE primer

<400>4

tgccaatcca gtggcaatag tgctga 26

<210>5

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223>5' RACE primer

<400>5

acaactgctg ccatcggaaa gggatt 26

<210>6

<211>2810

<212>DNA

<213> Tanggute white thorn

<400>6

acatggggaa acatcttcac ttgaaaatac ttccctctcc tctgtcttct gcctcttctc 60

tttttctttt tttttttttt cgttgttttt ctggttttgg taggtatggg agtggcgttg 120

ctgtccgagc tggcgacgga gatactggtt ccggtctgtg ccgtgatcgg tatcgtgttc 180

tcgctcgttc agtggtacct cgtctcgcgc gtgtcgctca cgcacgaccg gtcggccggg 240

aacaacaaca acaacaagaa gaatggattc aacgattatt tgatcgagga agaggaagga 300

attaatgacc agagcgtcgt gaccaagtgt gctgaaattc agaacgctat ttctgaaggt 360

gcaacatcct ttcttttcac tgaatatcag tatgttggga tcttcatggt tgcttttgca 420

atcttgattt tcctcttcct gggttctgtg gagggcttca gcacaaagag ccagcaatgt 480

acttacgata aaacaaggac gtgcaagcct gcacttgcca ctgctatctt cagcacagta 540

gcatttgtgc ttggtggcgt cacatctgtc ctttctggct tccttgggat gaaaattgct 600

acttatgcaa atgccagaac taccctggaa gcaagaaggg gtgtcggaaa ggcttttatt 660

actgcattta ggtctggtgc agtaatgggc ttcctccttg cagcaaatgg tctcttggtg 720

ctttacattg ctatcaatct ctttaagttg tactatggtg atgactggga aggcctattt 780

gaggctatta ctggatacgg tcttgggggt tcttcaatgg ctctctttgg acgagtgggt 840

ggtggtatct ataccaaggc tgctgatgtt ggtgctgatc ttgtgggcaa ggtcgagaga 900

aacattccag aagacgatcc aagaaaccct gctgtcattg ctgacaacgt tggtgacaat 960

gttggggaca ttgctggcat gggctcggat ctttttggtt cttatgctga gtcatcctgt 1020

gctgcgcttg ttgttgcatc catttcatcc tttggaatca accatgactt cactgccatg 1080

ttgtatcctc tgctcatcag ttcgatgggt atccttgttt gtttgatcac aactctcttt 1140

gccactgata tctttgagat caaggctgtt aaagagatcg agccagcatt gaagaagcag 1200

cttatcatct ctactattct tatgactgtt ggaattgcaa ttgtttcatg ggttggtttg 1260

ccttcgtcct tcacaaccta caattttggg actcagaagg ttgtcaagaa ctgggaactt 1320

ttcttgtgtg tgggtgttgg tctttgggct ggactcatca ttggatttgt gaccgagtac 1380

tatactagca atgcatacag ccctgtacaa gatgttgctg actcctgcag aacaggagct 1440

gccaccaatg ttatctttgg tcttgctttg ggatacaaat ctgttatcat tccaattttt 1500

gccattgcta tcagtatttt tgtcagtttt agtttggctg ctatgtatgg cattgcagtg 1560

gctgcccttg gtatgctcag cactattgcc actggattgg caattgatgc ctacggtccc 1620

atcagtgaca atgctggagg cattgctgag atggctggca tgagtcatcg catccgtgag 1680

aggactgatg cccttgatgc agctggaaac acaactgctg ccatcggaaa gggatttgca 1740

attggatcag cagcccttgt ctctttggca ctgtttggtg cttttgttag ccgtgcagca 1800

atctcaacag ttgatgtctt gactccaaag gtgttcatcg gtttgatcgt tggtgctatg 1860

cttccgtact ggttctctgc catgaccatg aagagtgcag gaagtgctgc attgaaaatg 1920

gttgaggagg ttcgcaggca gttcaacacc attcctggcc tcatggaggg cactgccaag 1980

cctgattacg ctaactgtgt caagatctct actgatgctt ccatcaagga gatgattcct 2040

cctggtgctc ttgtcatgct cacacccctc atcgtcggaa ccttcttcgg tgtggaaacc 2100

ctctctggtg ttcttgctgg ctctcttgta tctggtgttc agatcgcaat atctgcatca 2160

aacactggag gtgcatggga caatgccaag aagtacattg aggcaggtgc ttctgagcac 2220

gcaaggaccc ttggacccaa agggtcagag ccacacaagg cagctgtgat cggtgacacc 2280

atcggagacc cactcaagga cacgtcgggg ccatcactga acatccttat caagcttatg 2340

gccgtagaat cgcttgtttt tgctcccttt ttcgccaccc acggtgcctt tcttttcaaa 2400

attttttgaa gaaagttaaa taatgaccag aagaaggggg ggaagggagg gggtttgtta 2460

agctagtttt atttagggaa gttaaaatac tatttttgat tttgaggagg gatgaggagg 2520

aggatttgga caccaatgga gattggggta aaacaaaaat ttgatgggat gggatgtgtg 2580

aaccaaaatt ggaggcataa atatggggca tcaggacttg ggtgggttgg gggtatgttc 2640

tggtcctttt taattttaat ttttggtttt tttttttttt gtttcatgta gggggtagaa 2700

caattttccc ttttttattt tattttaaca tttgtactgt tcatcgttta atcaagaaaa 2760

gaccatttgg attttttacc aaaaaaaaaa aaaaaaaaaa aaaagtactt 2810

<210>7

<211>767

<212>PRT

<213> Tanggute white thorn

<400>7

Met Gly Val Ala Leu Leu Ser Glu Leu Ala Thr Glu Ile Leu Val Pro

1 5 10 15

Val Cys Ala Val Ile Gly Ile Val Phe Ser Leu Val Gln Trp Tyr Leu

20 25 30

Val Ser Arg Val Ser Leu Thr His Asp Arg Ser Ala Gly Asn Asn Asn

35 40 45

Asn Asn Lys Lys Asn Gly Phe Asn Asp Tyr Leu Ile Glu Glu Glu Glu

50 55 60

Gly Ile Asn Asp Gln Ser Val Val Thr Lys Cys Ala Glu Ile Gln Asn

65 70 75 80

Ala Ile Ser Glu Gly Ala Thr Ser Phe Leu Phe Thr Glu Tyr Gln Tyr

85 90 95

Val Gly Ile Phe Met Val Ala Phe Ala Ile Leu Ile Phe Leu Phe Leu

100 105 110

Gly Ser Val Glu Gly Phe Ser Thr Lys Ser Gln Gln Cys Thr Tyr Asp

115 120 125

Lys Thr Arg Thr Cys Lys Pro Ala Leu Ala Thr Ala Ile Phe Ser Thr

130 135 140

Val Ala Phe Val Leu Gly Gly Val Thr Ser Val Leu Ser Gly Phe Leu

145 150 155 160

Gly Met Lys Ile Ala Thr Tyr Ala Asn Ala Arg Thr Thr Leu Glu Ala

165 170 175

Arg Arg Gly Val Gly Lys Ala Phe Ile Thr Ala Phe Arg Ser Gly Ala

180 185 190

Val Met Gly Phe Leu Leu Ala Ala Asn Gly Leu Leu Val Leu Tyr Ile

195 200 205

Ala Ile Asn Leu Phe Lys Leu Tyr Tyr Gly Asp Asp Trp Glu Gly Leu

210 215 220

Phe Glu Ala Ile Thr Gly Tyr Gly Leu Gly Gly Ser Ser Met Ala Leu

225 230 235 240

Phe Gly Arg Val Gly Gly Gly Ile Tyr Thr Lys Ala Ala Asp Val Gly

245 250 255

Ala Asp Leu Val Gly Lys Val Glu Arg Asn Ile Pro Glu Asp Asp Pro

260 265 270

Arg Asn Pro Ala Val Ile Ala Asp Asn Val Gly Asp Asn Val Gly Asp

275 280 285

Ile Ala Gly Met Gly Ser Asp Leu Phe Gly Ser Tyr Ala Glu Ser Ser

290 295 300

Cys Ala Ala Leu Val Val Ala Ser Ile Ser Ser Phe Gly Ile Asn His

305 310 315 320

Asp Phe Thr Ala Met Leu Tyr Pro Leu Leu Ile Ser Ser Met Gly Ile

325 330 335

Leu Val Cys Leu Ile Thr Thr Leu Phe Ala Thr Asp Ile Phe Glu Ile

340 345 350

Lys Ala Val Lys Glu Ile Glu Pro Ala Leu Lys Lys Gln Leu Ile Ile

355 360 365

Ser Thr Ile Leu Met Thr Val Gly Ile Ala Ile Val Ser Trp Val Gly

370 375 380

Leu Pro Ser Ser Phe Thr Thr Tyr Asn Phe Gly Thr Gln Lys Val Val

385 390 395 400

Lys Asn Trp Glu Leu Phe Leu Cys Val Gly Val Gly Leu Trp Ala Gly

405 410 415

Leu Ile Ile Gly Phe Val Thr Glu Tyr Tyr Thr Ser Asn Ala Tyr Ser

420 425 430

Pro Val Gln Asp Val Ala Asp Ser Cys Arg Thr Gly Ala Ala Thr Asn

435 440 445

Val Ile Phe Gly Leu Ala Leu Gly Tyr Lys Ser Val Ile Ile Pro Ile

450 455 460

Phe Ala Ile Ala Ile Ser Ile Phe Val Ser Phe Ser Leu Ala Ala Met

465 470 475 480

Tyr Gly Ile Ala Val Ala Ala Leu Gly Met Leu Ser Thr Ile Ala Thr

485 490 495

Gly Leu Ala Ile Asp Ala Tyr Gly Pro Ile Ser Asp Asn Ala Gly Gly

500 505 510

Ile Ala Glu Met Ala Gly Met Ser His Arg Ile Arg Glu Arg Thr Asp

515 520 525

Ala Leu Asp Ala Ala Gly Asn Thr Thr Ala Ala Ile Gly Lys Gly Phe

530 535 540

Ala Ile Gly Ser Ala Ala Leu Val Ser Leu Ala Leu Phe Gly Ala Phe

545 550 555 560

Val Ser Arg Ala Ala Ile Ser Thr Val Asp Val Leu Thr Pro Lys Val

565 570 575

Phe Ile Gly Leu Ile Val Gly Ala Met Leu Pro Tyr Trp Phe Ser Ala

580 585 590

Met Thr Met Lys Ser Ala Gly Ser Ala Ala Leu Lys Met Val Glu Glu

595 600 605

Val Arg Arg Gln Phe Asn Thr Ile Pro Gly Leu Met Glu Gly Thr Ala

610 615 620

Lys Pro Asp Tyr Ala Asn Cys Val Lys Ile Ser Thr Asp Ala Ser Ile

625 630 635 640

Lys Glu Met Ile Pro Pro Gly Ala Leu Val Met Leu Thr Pro Leu Ile

645 650 655

Val Gly Thr Phe Phe Gly Val Glu Thr Leu Ser Gly Val Leu Ala Gly

660 665 670

Ser Leu Val Ser Gly Val Gln Ile Ala Ile Ser Ala Ser Asn Thr Gly

675 680 685

Gly Ala Trp Asp Asn Ala Lys Lys Tyr Ile Glu Ala Gly Ala Ser Glu

690 695 700

His Ala Arg Thr Leu Gly Pro Lys Gly Ser Glu Pro His Lys Ala Ala

705 710 715 720

Val Ile Gly Asp Thr Ile Gly Asp Pro Leu Lys Asp Thr Ser Gly Pro

725 730 735

Ser Leu Asn Ile Leu Ile Lys Leu Met Ala Val Glu Ser Leu Val Phe

740 745 750

Ala Pro Phe Phe Ala Thr His Gly Ala Phe Leu Phe Lys Ile Phe

755 760 765

<210>8

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> specific primer NtVP1-F8

<400>8

gttcgcaggc agttcaacac cattc 25

<210>9

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> specific primer NtVP1-R8

<400>9

accagataca agagagccag caaga 25

<210>10

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> UPM primer

<400>10

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210>11

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> T1 primer

<400>11

gtttgccttc gtccttca 18

<210>12

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> T2 primer

<400>12

cattgtccca tgcacctc 18

<210>13

<211>483

<212>DNA

<213> Artificial sequence

<220>

<223>207bp fragment

<400>13

gttcgcaggc agttcaacac cattcctggc ctcatggagg gcactgccaa gcctgattac 60

gctaactgtg tcaagatctc tactgatgct tccatcaagg agatgattcc tcctggtgct 120

cttgtcatgc tcacacccct catcgtcgga accttcttcg gtgtggaaac cctctctggt 180

gttcttgctg gctctcttgt atctggtgtt cagatcgcaa tatctgcatc aaacactgga 240

ggtgcatggg acaatgccaa gaagtacatt gaggcaggtg cttctgagca cgcaaggacc 300

cttggaccca aagggtcaga gccacacaag gcagctgtga tcggtgacac catcggagac 360

ccactcaagg acacgtcggg gccatcactg aacatcctta tcaagcttat ggccgtagaa 420

tcgcttgttt ttgctccctt tttcgccacc cacggtgcct ttcttttcaa aattttttga 480

aga 483

Claims

1. Tanggute white spine blister H⁺The gene NtVP1 of the PPase protein, and the full-length cDNA sequence of the gene is shown as SEQ ID NO. 6.

2. The Bursa of Thangodex sanguinea as claimed in claim 1⁺The gene NtVP1 of the PPase protein, wherein the sequence of the coding region of the gene is shown as 106-th and 2409-th positions of SEQ ID NO. 6.

3. Tanggute white spine blister H⁺The amino acid sequence of the PPase protein is shown as SEQ ID NO. 7.

4. Clone Tanggute spurge tonoplast H⁺-a method of the PPase protein gene NtVP1, comprising the steps of:

1) according to the vacuolar membrane H of the plant with the close relation with the Tanggute white thorn⁺-designing degenerate primer pairs for conserved regions of the PPase protein gene;

2) taking total RNA of the Nitraria tangutorum leaves as a template, and obtaining a cDNA fragment by using the degenerate primer pair of the step 1) through an RT-PCR method;

3) designing 3'RACE and 5' RACE primers according to the cDNA fragments obtained in the step 2), and obtaining a full-length cDNA sequence through nested amplification.

5. The method of claim 4, wherein the degenerate primer pair is NtVP1-F1 and NtVP 1-R1:

NtVP1-F1：CATTCGCCATTCAGG(SEQ ID NO:1)，

NtVP1-R1：ACATAGCAGCCAAAGT(SEQ ID NO:2)；

wherein, the cDNA segment with the sequence shown as SEQ ID NO. 3 is obtained in the step 2), and the length of the cDNA segment is 1156 bp;

in the step 3), the primer sequences are as follows:

UPM：

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT(SEQ ID NO:10)；

3’RACE：TGCCAATCCAGTGGCAATAGTGCTGA(SEQ ID NO:4)；

5’RACE：ACAACTGCTGCCATCGGAAAGGGATT(SEQ ID NO:5)；

wherein, the nucleotide sequence of the obtained full-length cDNA is shown as SEQ ID NO. 6.

6. An expression vector or expression strain comprising the Nitraria tangutorum bobr vacuole H according to claim 1 or 2⁺The PPase protein gene NtVP 1.

7. The expression vector or strain of claim 6, wherein the expression vector is a vector with GFP and the expression strain is Agrobacterium.

8. A method for constructing an expression vector comprising the Bursa of Tangut white Meadowrue H according to claim 1 or 2⁺-the PPase protein gene NtVP1, comprising: 1) xba I and Kpn I enzyme cutting sites are respectively added at two ends of a cDNA sequence of the NtVP1 gene or a coding region sequence thereof and are connected with a T vector to form a T-NtVP1 vector; 2) the T-NtVP1 vector, pCG-GFP and pSN1301 were digested with Xba I and Kpn I, respectively, and recovered; 3) the gene fragment cleaved from the T-NtVP1 vector was ligated to the cleaved fragments of the pCG-GFP and pSN1301 vectors.

9. A Chinese medicinal composition for treating acute leucorrhea with gastric ulcer⁺-a method for transfecting a plant with the PPase protein gene NtVP1 comprising transforming the expression vector or the expression strain of claim 6 into arabidopsis thaliana.

10. The Bursa of Thangodex sanguinea of claim 1 or 2⁺Application of the PPase protein gene NtVP1 in preparation or cultivation of transgenic plants.